The Putative Secreted Serine Protease Chp-7 Is Required for Full Virulence and Induction of a Nonhost Hypersensitive Response by Clavibacter michiganensis subsp. sepedonicus

Abstract

Molecular biological studies on Clavibacter michiganensis subsp. sepedonicus, the causal agent of bacterial ring rot of potato, have gained greater feasibility due to the recent availability of whole genomic sequences and genetic tools for related taxa. Here, we describe the first report of construction and characterization of a transposon (Tn) mutant library of C. michiganensis subsp. sepedonicus sp. strain R10. Since virulence of R10 in potato has been shown previously to be associated with elicitation of a nonhost hypersensitive response (HR), the mutant library was screened initially for loss of HR in tobacco. The screen identified two HR-negative mutants containing Tn insertions within the same gene, CMS2989 (chp-7), although at distinct locations. chp-7 is one of 11 pat-1 homologs in C. michiganensis subsp. sepedonicus. HR-negative mutants of R10 multiplied to the same extent as wild type in planta but were less virulent in potato. Complementation with chp-7 restored virulence as well as the HR phenotype. Together, these findings demonstrate a role for chp-7 in C. michiganensis subsp. sepedonicus-plant interactions.

Putative Serine Protease Effectors of Clavibacter michiganensis Induce a Hypersensitive Response in the Apoplast of Nicotiana Species

“…The chp-7 S232T derivative was made by PCR using KOD Hot Start DNA polymerase (Novagen, CA) using pRLSTC7 (Syverson 2011) as template with primers C7_S232T_F and C7_S232T_R followed by self-ligation with T4 DNA ligase (New England Biolabs, MA), resulting in pRLSTC7ST. Both plasmids were transformed into competent cells of CmsC7X20 by electroporation at 1.5 kV in cuvettes with a 1 mm gap width as previously described (Nissinen et al 2009). C-terminal FLAG epitope tags were inserted into pHNC7 and pHNC7ST by PCR with primers C7_OLpHN_F, C7_FLAG_R and FLAG_OLpHN_R, followed by ligation using the Gibson assembly cloning kit (New England Biolabs, MA) into pHN216 cut open with EcoRI, resulting in pHNC7-F and pHNC7ST-F.…”

“…One close homolog of pat-1, named chp-7 (CMS_2989), has been identified as an important virulence factor of Cms required for full symptom development in infected potato and eggplant but not for colonization (Nissinen et al 2009). Like Pat-1, Chp-7 contains a predicted catalytic histidine at position 97 within a TAKHC motif and the predicted nucleophilic serine at position 232 within a GDSGG motif (Nissinen et al 2009). Though no proteolytic activity has been demonstrated for any of the pat-1 family serine proteases, the serine protease activity of Pat-1 is thought to be involved in virulence of Cmm in tomato, as exchanging the putative catalytic serine for threonine in Pat-1 rendered it unable to restore the virulence of the plasmid-free strain CMM100 (Burger et al 2005).…”

“…covalent attachment to the cell wall of Gram-positive bacteria(Burger et al 2005, Nissinen et al 2009). However, the detection of Chp-7-FLAG in the culture supernatant by immunoblot with monoclonal anti-FLAG antibody suggests the Cterminal region downstream of the LPGSG motif was not cleaved by the Cms sortase.…”

Molecular characterization of a serine protease Pro1 from Plasmodiophora brassicae that stimulates resting spore germination

“…As a pathogenicity factor, serine protease has been reported from various pathosystems (Dow et al, 1990;Dreier et al, 1997;Nissinen et al, 2009;Redman and Rodriguez, 2002). Dow et al (1990) suggested that the role of bacterial proteases may be nutritional or, alternatively, that they could aid in the infection process through the proteolysis of structural proteins in plant cell walls.…”

“…In the potato ring rot pathogen C. michiganensis ssp. sepedonicus , a secreted serine protease Chp‐7 has been identified and proven to be required for full virulence and induction of a nonhost hypersensitive response (Nissinen et al ., 2009). Redman and Rodriguez (2002) demonstrated that an extracellular serine protease is required for the pathogenicity of Colletotrichum coccodes , and that the elimination of protease activity transforms a virulent pathogen to an avirulent endophyte.…”